Albumin is a protein naturally found in the blood plasma of mammals, where it is the most abundant protein. It has important roles in maintaining the desired osmotic pressure of the blood and also has a role in the transport of various substances within the blood stream.
Albumin binds in vivo to its receptor, the neonatal Fc receptor (FcRn) “Brambell” and this interaction is known to be important for increasing the plasma half-life of albumin. FcRn is a membrane bound protein, expressed in many cell and tissue types, and has been found to reduce the rate of intracellular degradation of albumin (Roopenian, D. C. and Akilesh, S. (2007), Nat. Rev. Immunol 7, 715-725.). FcRn contributes to maintaining both the high level of IgG and albumin in the serum of mammals, such as human beings.
Whilst the FcRn-immunoglobulin G (IgG) interaction has been characterized in the prior art, the FcRn-albumin interaction is less well characterized or understood. The major albumin-FcRn binding-site is localized within domain III (DIII: 381-585) (Andersen et al (2010) Clinical Biochemistry 43, 367-372). However, it is known within the art that both IgG and albumin bind non-cooperatively to distinct sites on FcRn (Andersen, et al. (2006), Eur. J. Immunol 36, 3044-3051; Chaudhury, et al. (2006), Biochemistry 45, 4983-4990.).
Human serum albumin (HSA) has been characterized as a polypeptide of 585 amino acids, the sequence of which can be found in Peters, T., Jr. (1996) All about Albumin: Biochemistry, Genetics and Medical, Applications, pp 10, Academic Press, Inc., Orlando (ISBN 0-12-552110-3). Albumin has a characteristic pH-dependent binding to FcRn, where it binds at an acidic pH, such as pH 6.0 but not at a pH above neutral, such as pH 7.4.
The plasma half-life of HSA has been found to be approximately 19 days (Peters, T., Jr. (1985) Adv. Protein Chem. 37, 161-245; Peters, T., Jr. (1996) All about Albumin, Academic Press, Inc., San Diego, Calif. (page 245-246)); Benotti P, Blackburn GL: Crit Care Med (1979) 7:520-525). A naturally occurring point mutant, having the substitution D494N has a lower plasma half-life (Biochim Biophys Acta. 1991, 1097:49-54). This single substitution creates an N-linked glycosylation site in this variant/mutant, which is not present in wild-type (wt) HSA. It is not known whether the potential glycosylation at this site or the amino acid change itself is responsible for the change in plasma half-life observed.
Albumin is a plasma protein considered to have a long plasma half-life and because of this property it has been suggested to be used in drug delivery. Albumin has been conjugated to pharmaceutically beneficial compounds (WO0069902A). Hence, the resultant plasma half-life of the conjugates has generally been found to be considerably longer than the plasma half-life of the beneficial compounds alone.
Further, albumin has been fused to therapeutically beneficial peptides (WO 01/79271 A and WO 03/59934 A), with the typical result that the fusion polypeptides have the activities of the therapeutically beneficial peptides and a long plasma half-life, which is considerably greater than the plasma half-life of the therapeutically beneficial peptides alone.
Otagiri et al (2009), Biol. Pharm, Bull. 32(4), 527-534, discloses that 77 albumin variants are known, of these 25 are found in domain III. A natural variant lacking the last 175 amino acids at the carboxy terminus has been shown to have reduced half-life (Andersen et al (2010), Clinical Biochemistry 43, 367-372). Iwao et al. (2007) studied the half-life of naturally occurring human albumin variants using a mouse model, and found that K541E and K560E had reduced half-life, E501K and E570K had increased half-life and K573E had almost no effect on half-life (Iwao, et. al. (2007) B.B.A. Proteins and Proteomics 1774, 1582-1590).
Galliano et al (1993) Biochim. Biophys. Acta 1225, 27-32 discloses a natural variant E505K. Minchiotti et al. (1990) discloses a natural variant K536E. Minchiotti et al (1987) Biochim. Biophys. Acta 916, 411-418 discloses a natural variant K574N. Takahashi et al (1987) Proc. Natl. Acad. Sci. USA 84, 4413-4417, discloses a natural variant D550G. Carlson et al (1992). Proc. Nat. Acad. Sci. USA 89, 8225-8229, discloses a natural variant D550A.
WO 2007112940 discloses constructs comprising at least one albumin domain III and at least one therapeutic moiety and the use of such constructs for half-life extension of drugs.
Albumin has the inherent ability to allow the binding of a number of ligands, and these become associated (associates) with albumin. This property has been utilized to extend the plasma half-life of such aforementioned ligands, e.g. to extend the plasma half-life of drugs having the ability to non-covalently bind to albumin. This can also be achieved by binding a pharmaceutically beneficial compound, which has little or no albumin binding properties, to a moiety having albumin-binding properties. See review article and reference therein: Kratz (2008). Journal of Controlled Release 132, 171-183.
U.S. Pat. No. 7,253,259 discloses a protein produced by gene recombinant technology including at least one domain selected from domains I, II and III of serum albumin but having a different structure from that of native albumin; and a method of producing the protein.
Albumin is used in preparations of pharmaceutically beneficial compounds, in which such a preparation maybe for example, but not limited to, a nano particle or micro particle of albumin. In these examples the delivery of a pharmaceutically beneficial compound or mixture of compounds may benefit from alteration in the albumins affinity to the FcRn receptor where the beneficial compound has been shown to associate with albumin for the means of delivery.
The exact nature or associated properties that influences the extension to the plasma half-life of the formed conjugates or fusion polypeptides is unclear (for example, but not limited to, Levemir®, Kurtzhals P et al. Biochem. J. 1995; 312:725-731), but it appears to be directly related to the albumin moiety and the selected pharmaceutically beneficial compound/peptide they are composed of. It would be desirable to be able to control the plasma half-life of a given albumin domain III derivative, fragments, or variants thereof with a conjugated or fusion or association such that a longer or shorter plasma half-life, than given by the components of the conjugate/fusion alone, can be achieved. This would allow the custom design of a particular drug according to the particulars of the indication intended to be treated.
Albumin is known to accumulate and be catabolised in tumours, it has also been shown to accumulate in inflamed joints of rheumatoid arthritis sufferers. See review article and reference therein, Kratz (2008). Journal of Controlled Release 132, 171-183. It is envisaged that HSA variants with increased affinity for FcRn would be advantageous for the delivery and/or targeting (such as passive targeting) of pharmaceutically beneficial compounds.
It may be desirable to have variants of albumin that have little or no binding to FcRn in order to provide shorter half-lives or controlled serum pharmacokinetics as described by Vania Kenanova, Tove Olafsen, Felix Bergara and Anna Wu (2009) J Nucl Med.; 50 (Supplement 2):1582).